植物细胞抑制素是普遍存在于植物和动物中的半胱氨酸蛋白酶抑制剂。已知它们执行各种重要的生理功能并且还维持蛋白酶-抗蛋白酶活性的平衡。在目前的研究中,通过简单的两步策略,使用硫酸铵分馏和在SephacrylS-100-HR上进行的凝胶过滤色谱,将经过初步处理的植物胱抑素从大豆(Glycinemax)中分离并纯化至均质。以635倍纯化和77.6%的%产率纯化大豆植物胱抑素(SBPC)。在天然凝胶电泳上观察到单条带,证实纯化的SBPC的均一性。经SDS-PAGE测定,SBPC的分子量为19.05kDa。发现SBPC缺乏碳水化合物部分和巯基含量。通过等温量热法确定SBPC-木瓜蛋白酶相互作用的结合化学计量,提示1:1复合物,并且发现结合常数(K)的值为2.78×105M-1。通过ITC获得的结合亲和力(Kd)值为3.59×10-6M。发现纯化的SBPC在pH范围内稳定3至7,并且在高达50°C的温度下是稳定的。紫外可见和荧光研究表明,SBPC-木瓜蛋白酶复合物形成后,构象发生了显着变化。此外,荧光光谱法,ANS绑定,并进行了酪蛋白分解活性测定,以探讨金属离子对SBPC的影响,结果表明,与Cd2和Ni2形成络合物时,SBPC的抑制活性随构象变化而丧失。
Phytocystatins are cysteine proteinase inhibitors ubiquitously present in plants and animals. They are known to carry out various significant physiological functions and also maintain the balance of protease-antiprotease activity. In the present disquisition, a
phytocystatin after preliminary treatment has been isolated and purified to homogeneity from soybean (Glycine max) by a simple two-step stratagem using ammonium sulfate fractionation and gel filtration chromatography performed on Sephacryl S-100-HR. Soybean
phytocystatin (SBPC) was purified with a fold purification of 635 and percent yield of 77.6%. A single band was observed on native gel electrophoresis confirming the homogeneity of the purified SBPC. The molecular weight of SBPC was found to be 19.05 kDa as determined by SDS-PAGE. The SBPC was found to be devoid of carbohydrate moieties and sulfhydryl group content. The binding stoichiometry of SBPC-papain interaction was determined by isothermal calorimetry suggesting 1:1 complex, and the value of binding constant (K) was found to be 2.78 × 105 M-1 The affinity of binding (Kd ) value obtained through ITC was 3.59 × 10-6 M. The purified SBPC was found to be stable in the pH range of 3 to 7 and is thermostable up to 50°C. The UV-visible and fluorescence studies showed significant changes in the conformation upon the formation of the SBPC-papain complex. Furthermore, fluorescence spectroscopy, ANS binding, and caseinolytic activity assay were conducted out to explore the effect of metal ions on SBPC which showed that there was a loss in the inhibitory activity along with conformational changes of SBPC upon complex formation with Cd+2 and Ni+2 .